1. Field of the Invention
This invention relates to a novel microorganism, and methods for biodegradation of organic compounds and for environmental remediation.
2. Related Background Art
Environmental pollution with hardly decomposable organic compounds that are toxic for organisms has become a serious problem in recent years. It is thought that pollution of soil with volatile chlorinated aliphatic hydrocarbon compounds such as tetrachloroethylene (PCE), trichloroethylene (TCE) and dichloroethylene (DCE) has been spreading considerably at the sites of paper and pulp industries, precession instrument industries and related industries in the world. Actually, it is often reported that these pollutants were detected through environmental surveys. It is supposed that these residual organic compounds in the soil are dissolved into rainwater and permeate into ground water, thereby spreading over surrounding area. Since these compounds are suspected to be carcinogenic besides being quite stable in the environment, contamination of ground water that is used as a source of drinking water especially has become a serious social problem. Therefore, cleaning of aqueous media such as contaminated ground water, and the soil and surrounding atmosphere by eliminating or decomposing polluting organic compounds is an important theme for environmental protection, and technologies required for cleaning the environment have been developed. For example, an absorption treatment with activated carbon and decomposition treatment by light or heat have been studied, but they are not always practical from the viewpoint of treatment cost and operability.
Recently, microbial decomposition of chlorinated organic compounds like TCE which are stable in the environment has been reported, and studies for its practical application have been started. The biodegradation method has several advantages that halogenated organic compounds can be decomposed into harmless substances by selecting appropriate microorganisms, specific chemicals are not needed and the maintenance cost and labor for the process can be saved.
Examples of microorganisms capable of decomposing TCE are Welchia alkenophila sero 5 (U.S. Pat. No. 4,877,736, ATCC 53570), Welchia alkenophila sero 33 (U.S. Pat. No. 4,877,736, ATCC 53571), Methylocystis sp. strain M (Agric Biol. Chem., 53, 2903 (1989), Biosci. Biotech. Biochem., 56, 486 (1992), ibid 56, 736 (1992)), Methylosinus trichosporium OB3b (Am. Chem. Soc. Natl. Meet. Dev. Environ. Microbiol., 29, 365 (1989), Appl. Environ. Microbiol., 55, 3155 (1989), Appl. Biochem. Biotechnol., 28, 877 (1991), Japanese Laid-Open Patent Application No. 2-92274, Japanese Laid-Open Patent Application No. 3-292970), Methylomonas sp. MM2 (Appl. Environ. Microbiol., 57, 236 (1991)), Alcaligenes denitrificans ssp. xylosoxidans JE75 (Arch. Microbiol., 154, 410 (1990)), Alcaligenes eutrophus JMP134 (Appl. Environ. Microbiol., 56, 1179 (1990)), Alcaligenes eutrophus FERM-13761 (Japanese Laid-Open Patent Application No. 7-123976), Pseudomonas aeruginosa JI104 (Japanese Laid-Open Patent Application No. 7-236895), Mycobacterium vaccae JOB5 (J. Gen. Microbiol., 82, 163 (1974), Appl. Environ. Microbiol., 55, 2960, (1989), ATCC 29678), Pseudomonas putida BH (J. of Japan Sewage Work Assoc. (Gesuido Kyokai-shi), 24, 27 (1987), Pseudomonas sp. strain G4 (Appl. Environ. Microbiol., 52, 383, (1986), ibid 53, 949 (1987), ibid 54, 951 (1989), ibid 56, 279 (1990), ibid 57, 193 (1991), U.S. Pat. No. 4,925,802, ATCC 53617, this strain was first classified as Pseudomonas cepacia but later changed to Pseudomonas sp.), Pseudomonas mendocina KR-1 (Bio/Technol., 7, 282 (1989), Pseudomonas putida F1 (Appl. Environ. Microbiol., 54, 1703 (1988), ibid 54, 2578 (1988)), Pseudomonas fluorescens PFL12 (Appl. Environ. Microbiol., 54, 2578 (1988)), Pseudomonas putida KWI-9 (Japanese Laid-Open Patent Application No. 6-70753), Pseudomonas cepacia KK01 (Japanese Laid-Open Patent Application No. 6-22769), Nitrosomonas europaea (Appl. Environ. Microbiol., 56, 1169 (1990)) and Lactobacillus vaqinalis sp. nov (Int. J. Syst. Bacteriol., 39, 368 (1989), ATCC 49540).
The problem in practically applying these decomposing bacteria for remedying the environment is that they require chemicals such as aromatic compounds or methane as an induction substance (an inducer).
Although aromatic compounds such as phenol or toluene are very excellent inducers, an intensive control is necessary when these compounds are discharged into the environment because they have toxicity. Methane is a flammable gas so that there is danger and difficulty in introducing it into the environment with control.
To solve the problems described above, Nelson et. al. developed a method to use tryptophan, an amino acid as an inducer for decomposition of volatile chlorinated organic compounds (Japanese Laid-Open Patent Application No. 4-502277). Although this method can avoid the toxicity and danger of the inducer itself to some extent, tryptophan is a very expensive compound, and the difficulty of introducing a specific substance into the environment with control is not still solved. Adding excess carbon and nitrogen source into the environment is also not preferable from the view point of eutrophication. Furthermore, since such TCE decomposition enzymes are inducible enzymes, the enzymatic activity once induced is usually sustained for from only several hours to a day, requiring another induction after that, and there is a problem that decomposition of TCE is competitively inhibited by the presence of the inducing agents.
Recently, there has been attempted to introduce an plasmid having a DNA fragment encoding oxygenase or hydroxylase as a TCE decomposition enzyme into a host bacterium in order to express the TCE decomposition activity using a harmless inducer or to constitutively express it in the absence of any inducers. For example, such DNA fragments are derived from Pseudomonas mendocina KR-1 (Japanese Laid-Open Patent Application No. 2-503866), Pseudomonas putida KWI-9 (Japanese Laid-Open Patent Application No. 6-105691) and Pseudomonas putida BH (Summary of 3rd Conference on Pollution of Ground Water/Soil and Its Protective Countermeasure, p.213 (1994).
These recombinant strains, however, have such problems that a very expensive IPTG (isopropyl thiogalactopyranoside) is required as an inducer or stability of the plasmid in the host bacterium strain is not sufficient. Moreover, the discharge of recombinant strains into the environment is under certain regulations considering the public acceptance.
To solve these problems, Seals et. al. obtained a mutant strain of Pseudomonas sp. G4 having a TCE decomposition activity without requiring any inducer (phenol or toluene in this case) by a method using a transposon (Appl. Environment. Microbiol., 58, 3977 (1992), PCT Application, International Publication No. WO 92/19738). The TCE decomposition activity of this G4 mutant is, however, is not sufficient apart from the problem of instability due to the transposon. Since the transposon itself contains an antibiotic resistance gene such as kanamycin resistance, there may be some undesirable effects by a horizontal transmission to other microorganisms when the mutant strain is released into the environment.
As a countermeasure of this problem, it is reported in Environmental Science & Technology, Vol. 30, No. 6, 1986, pp. 2045-2052 that a mutant, Burkholderia (Pseudomonas) cepacia PR1.sub.301 (denoted as "PR1.sub.301 " hereinafter), which can decompose TCE without inducers, was obtained by mutagenizing the above strain G4 using nitrosoguanidine instead of genetic manipulation. However, according to the data of the above report, for example, FIG. 3 in the right column in page 2048 or FIG. 7 in the left column in page 2050, the TCE degradation ability of PR1.sub.301 is in ppb order, insufficient for the remediation of the environment polluted with TCE.
As hitherto described, the bacterial species known in the art are not always satisfactory from the viewpoint that a microorganism not requiring inducers should satisfy practical conditions required for the decomposition of aromatic compounds or volatile chlorinated organic compounds and should express a sufficient decomposition ability. Therefore, there is an urgent need of obtaining microorganisms having practically required characteristics. Preferably, these microorganisms have such properties that they have a sufficient ability to degrade a pollutant such as aromatic compounds and halogenated aliphatic hydrocarbon compounds, and their growth conditions are different from the conventional microorganisms known in the art so that their application range or application form can be more expanded. During the treatment of liquid wastes or soil containing TCE using a microorganism, it is required for the microorganism to proliferate under poor conditions such as liquid waste or soil, while maintaining the required TCE-degrading activity.